Topical microbicides are defined as vaginally applied products that prevent male-to-female or female-to-male HIV transmission. The high error rate of the HIV reverse transcriptase (RT) drives the development of resistance and genomic diversity in HIV. The current pan-resistance to all classes of HIV inhibitors such as that found in O group virus for non-nucleoside RT and protease (PR) inhibitors, raises an important question with regard to the development of microbicides targeting RT or PR. These concerns may be extended to inhibitors of HIV entry due to rapid emergence of mutations in the envelope (Env) under selective pressure. There is thus an urgent need to identify new anti-HIV compounds, which target viral components other than PR, RT and Env, and which may be developed as safe and effective microbicides. We identified a short linear peptide, which neutralizes HIV at an nM range. The peptide blocks HIV entry into cells in an Env-independent manner. Several lines of evidence suggest that the peptide destabilizes the HIV membrane, liberating the Env glycoprotein. The peptide-mediated HIV membrane destabilization is specific because i) the peptide does not disturb the membrane of other enveloped viruses (i.e., HSV and VSV); ii) it does not destabilize the membrane of human cervical cells even when applied twice daily to cells at a concentration 20-200-fold superior to that which blocks HIV infection; iii) the peptide does not harm mice injected i.v. twice daily with high peptide concentrations (0.5 mg) for a period of three days; and iv) the peptide apparently does not create lesions in the vaginal epithelium of humanized mice since the peptide, rather than promoting transmission, completely blocks intravaginal transmission The peptide represents an attractive microbicide candidate for several reasons: i) the peptide inhibits infection of a broad range of primary isolates in various primary human cell types; ii) it interferes with the three mechanisms involved in HIV transmission: genital epithelial transmigration, dendritic cell-mediated transmission, and infection of mucosal target cells; iii) the peptide is extremely efficacious since less than 15 minutes of exposure suffices to neutralize HIV; iv) it is potent for two hours both prior to and after addition of HIV to cells, suggesting that it should be active for an acceptable length of time both prior to and after sexual intercourse; v) the peptide retains its antiviral properties at a low pH; vi) it is not toxic or immunogenic in mice, even after repeated i.v. administrations of high peptide doses; and vii) most importantly, the peptide totally blocks the intravaginal transmission of HIV in humanized mice. In this application, we propose to fully characterize the anti-HIV efficacy of the peptide and derivatives in vitro as well as their toxicity and immunogenicity in vivo (R21 phase). If these studies unambiguously demonstrate that the peptides represent attractive anti-HIV microbicide candidates, they will then be tested as topical microbicides in vivo (R33 phase). The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of short peptides as novel anti-HIV microbicides. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV-1 could play a major role in worldwide reduction of the over 14,000 new HIV-1 infections per day, and potentially save millions of lives.